团头鲂hepcidin基因单核苷酸多态性及与抗病性状的相关性
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摘要
为了解hepcidin(铁调素)基因的多态性及其与抗病性状的相关性,以团头鲂(Megalobrama amblycephala)为研究对象,从公共数据库中获得hepcidin基因序列,对团头鲂进行嗜水气单胞菌(Aeromonas hydrophila)人工感染来区分易感和抗病个体,采用PCR扩增及测序技术筛选hepcidin基因序列中的SNP(单核苷酸多态,single nucleotide polymorphisms)位点,对SNP位点运用高分辨率熔解曲线法(high-resolution melting,HRM)进行分型,分析其多态性及与抗病性状之间的关系。结果在hepcidin基因上共筛选出2个SNP位点,其中1个位点位于内含子部分,1个位于3′非编码区。经过统计分析发现,位于3 371bp(A/G)的这一SNP位点的基因型频率和等位基因频率在易感和抗病组间差异极显著,位于217bp(A/G)位点的基因型频率和等位基因频率在易感组和抗病组间差异显著。据此推测hepcidin的这2个SNP位点多态性与团头鲂抗病性状相关。
The genomic sequences of hepcidin in Megalobrama amblycephala were obtained from public database to study the association between the hepcidin gene polymorphism and disease resistance trait.Aeromonas hydrophilachallenge experiment was used to distinguish susceptible and disease resistant M.amblycephalaindividuals.The single nucleotide polymorphisms(SNP)of the hepcidin gene in M.amblycephala were screened with PCR and DNA sequencing.High-resolution melting(HRM)were used for SNP genotyping.Two SNPs of the hepcidin gene were screened,one of which was located in the intron region and the other was located in the 3′-untranslated region.The results of statistical analyses showed that the genotype and allele frequencies of SNP(A/G)located at 3 371 bp was extremely significant different(P<0.01)and SNP(A/G)located at 217 bp was significant different(P<0.05)between the susceptible and resistant group.It is indicated that the polymorphism of hepcidin gene was significantly associated with bacterial septicemia resistance.
The genomic sequences of hepcidin in Megalobrama amblycephala were obtained from public database to study the association between the hepcidin gene polymorphism and disease resistance trait.Aeromonas hydrophilachallenge experiment was used to distinguish susceptible and disease resistant M.amblycephalaindividuals.The single nucleotide polymorphisms(SNP)of the hepcidin gene in M.amblycephala were screened with PCR and DNA sequencing.High-resolution melting(HRM)were used for SNP genotyping.Two SNPs of the hepcidin gene were screened,one of which was located in the intron region and the other was located in the 3′-untranslated region.The results of statistical analyses showed that the genotype and allele frequencies of SNP(A/G)located at 3 371 bp was extremely significant different(P<0.01)and SNP(A/G)located at 217 bp was significant different(P<0.05)between the susceptible and resistant group.It is indicated that the polymorphism of hepcidin gene was significantly associated with bacterial septicemia resistance.
引文
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[30]BAO B,PEATMAN E,LI P,et al.Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection[J].Dev Comp Immunol,2005,29(11):939-950.
[31]CHEN S L,LI W,MENG L,et al.Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot(Scophthalmus maximus)[J].Fish Shellfish Immunol,2007,22(3):172-181.
[32]BARNES A C,TREWIN B,SNAPE N,et al.Two hepcidinlike antimicrobial peptides in Barramundi latescalcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide[J].Fish Shellfish Immunol,2011,31(2):350-357.
[2]柯鸿文.中国团头鲂的养殖[J].水产科技情报,1986(5):1-4.
[3]王卫民.团头鲂养殖产业现状[J].科学养鱼,2009(4):49-50.
[4]农业部渔业局.中国渔业统计年鉴[M].北京:中国农业出版社,2018:45.
[5]姚伦,王玉兰,涂彭文.团头鲂爆发性败血症病原分离鉴定及免疫防治[J].江西水产科技,2008(4):16-19.
[6]王春瑞.嗜水气单胞菌的分子流行病学调查及其两种分型方法的研究[D].宁波:宁波大学,2011.
[7]杜玮南,孙红霞,方福德.单核苷酸多态性的研究进展[J].中国医学科学院学报,2000,22(4):392-394.
[8]潘登.高分辨率熔解曲线分析技术(HRM)系统的建立及其在基因多态性和突变检测方面的应用[D].上海:复旦大学,2013.
[9]王卫卫,吴谡琦,孙修勤,等.硬骨鱼免疫系统的组成与免疫应答机制研究进展[J].海洋科学进展,2010,28(2):257-265.
[10]常彦忠,段相林,钱忠明.Hepcidin和铁稳态[J].中华内分泌代谢杂志,2003,19(6):501-504.
[11]LIANG T,JI W,ZHANG G R,et al.Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1(LEAP-1)and LEAP-2genes in the blunt snout bream(Megalobrama amblycephala)[J].Fish Shellfish Immunol,2013,35(2):553-563.
[12]BECK V B S,SCHULER M A,JOLLIE D R,et al.Synthesis,bacterial expression,and mutagenesis of the gene coding for mammalian cytochrome b5[J].Proc Natl Acad Sci USA,1986,83(24):9443-9447.
[13]杨明.养殖真鲷、黑鲷抗菌肽hepcidin的基因克隆、表达特性及其抗菌活性研究[D].厦门:厦门大学,2006.
[14]高宇,陈昌福,李大鹏,等.中华鲟抗菌肽Hepcidin的克隆、表达及其抗菌活性分析[J].水生生物学报,2012,36(4):798-803.
[15]周凤娟.团头鲂NLRC3-like基因的克隆、表达及其SNP的开发和鉴定[D].武汉:华中农业大学,2015.
[16]柴欣,胡晓坤,马徐发,等.团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析[J].华中农业大学学报,2017,36(4):76-82.
[17]LIU H,CHEN C,GAO Z,et al.The draft genome of blunt snout bream(Megalobrama amblycephala)reveals the development of intermuscular bone and adaptation to herbivorous diet[J].Giga science,2017,6(7):1-13.
[18]柴欣.团头鲂MHCⅡα基因的SNP位点开发、鉴定及与抗病性状关联分析[D].武汉:华中农业大学,2017.
[19]黄文树,李少菁,蔡灵,等.尼罗罗非鱼Hepcidin基因结构与序列分析[J].厦门大学学报(自然科学版),2007,46(3):390-395.
[20]李华.鲤鱼抗菌肽hepcidin的基因克隆、表达及其功能的研究[D].济南:山东师范大学,2010.
[21]XU T J,CHEN S L,ZHANG Y X.MHC classⅡalpha gene polymorphism and its association with resistance/susceptibility to Vibrio anguillarumin Japanese flounder(Paralichthys olivaceus)[J].Dev Comp Immunol,2010,34(10):1042-1050.
[22]LOHM J,GRAHN M,LANGEFORS A,et al.Experimental evidence for major histocompatibility complex-allele-specific resistance to a bacterial infection[J].Proc R Soc B,2002,269(1504):2029-2033.
[23]SAEIJ J P,STET R J,DE VRIES B J,et al.Molecular and functional characterization of carp TNF:a link between TNF polymorphism and trypanotolerance[J].Dev Comp Immunol,2003,27(1):29-41.
[24]衡建福.草鱼TLR3和TLR22基因多态性与草鱼出血病抗性之间的关联分析[D].杨凌:西北农林科技大学,2011.
[25]万全元.草鱼RIG-I基因核苷酸多态性与草鱼呼肠孤病毒感染的关联研究[D].杨凌:西北农林科技大学,2014.
[26]王兰.草鱼Mx2和MDA5基因多态性与草鱼出血病抗性的关联研究[D].杨凌:西北农林科技大学,2012.
[27]黄新芳,沈南,梁栋,等.PBX1基因3′末端基因多态性rs3185695与系统性红斑狼疮发病相关研究[J].中华风湿病学杂志,2006,10(3):129-132.
[28]KULAKSIZ H,THEILIG F,BACHMANN S,et al.The ironregulatory peptide hormone hepcidin:expression and cellular localization in the mammalian kidney[J].J Endocrinol,2005,184(2):361-370.
[29]SHIKE H,SHIMIZU C,LAUTH X,et al.Organization and expression analysis of the zebrafish hepcidin gene,an antimicrobial peptide gene conserved among vertebrates[J].Dev Comp Immunol,2004,28(7):747-754.
[30]BAO B,PEATMAN E,LI P,et al.Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection[J].Dev Comp Immunol,2005,29(11):939-950.
[31]CHEN S L,LI W,MENG L,et al.Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot(Scophthalmus maximus)[J].Fish Shellfish Immunol,2007,22(3):172-181.
[32]BARNES A C,TREWIN B,SNAPE N,et al.Two hepcidinlike antimicrobial peptides in Barramundi latescalcarifer exhibit differing tissue tropism and are induced in response to lipopolysaccharide[J].Fish Shellfish Immunol,2011,31(2):350-357.